INTRODUCTION:

In today's, medicinal plants have captured a significant view due to their special property as large source of phytochemicals that are therapeutically important that may lead to the development of new drugs. To know more about the medicinal plants, it is important to the study the details account of medicinal plants and that can start with the pre-extraction which includes the preparation of a plant sample with respect to its drying, grinding etc. and extraction methodology, which is one of the important steps to isolate the therapeutically active phytochemical from the medicinal plants. This isolated phytochemical serves as starting material for the development or production of biologically active compounds.

With an advantage of pre-extraction and extraction methodology, selection of suitable extraction methodology and optimization of various critical parameters for the suitable extraction method is also important criteria for the standardization with some up-scaling purposes i.e. from lab scale to pilot scale requires in the development of the therapeutically active compound^[1].

Various extraction methodology most commonly used mainly for the extraction of active compound from the medicinal plant material includes conventional extraction (CEM) and non-conventional extraction methodology (N-CEM). The conventional extraction methodology is simple and mostly includes Maceration, Percolation, Infusion, Decoction etc. on the other hand; the non-conventional methodology includes Ultrasound Assisted Extraction (UAE), Microwave-Assisted Extraction (MAE), Accelerated Solvent Extraction (ASE) and Supercritical Fluid Extraction (SFE). Recently, this N-CEM has gained increasing interest and has been rapidly spread globally for the isolation of therapeutically important phytochemical from the medicinal plants as this N-CEM has an advantage over the CEM. The N-CEM is fast and environmentally friendly in terms of solvents and energy consumptions and several parameters can be controlled at a time. Furthermore, improvement in the extraction efficiency, efficacy, batch to batch variation, consistency and selectivity are also the benefits of the N-CEM^[2-4]. After the extraction of medicinal plants by CEM and N-CEM, the estimation of bioactive can be done by HPLC. High performance liquid Chromatography (HPLC) is an important analytical tool for quantification of bioactive compound in herbal extract. It is commonly used chromatographic technique for the estimation of secondary metabolites in the plants. It has wide applications in different fields in term of isolation, qualitative and quantitative estimation of active molecules^[5-13].

Baicalein (5, 6, 7-trihydroxyflavone) is one of the phytochemical is a flavone, a type of flavonoid, originally obtained from roots of Scutellaria baicalensis and Scutellaria lateriflora (Structure of Baicalein shown in figure 1). It is also reported in Oroxylum indicum or Indian trumpet flower belonging to family Bignoniaceae. It is also known as Sonapatha or Shyonaka. It is a commonly used herbal medicine in an Ayurvedic system ^[14]. Roots, leaves, and stems of O. indicum have been used as a single drug or as a component of certain compound drug preparations in the Indian Ayurvedic system of medicine for treatment of various disorders as well as used as a tonic and Rasayana drug. It contains flavonoid like chrysine, baicalein, and Oroxylin-A. Various studies indicated that sonapatha possesses anti-inflammatory, anticancer, antioxidant, hepatoprotective, antimutagenic and immunomodulatory properties^[15-24]. The root bark of O. indicum possesses antiulcer activity^[25].

Figure 1- Chemical Structure of Baicalein

Baicalein has historically been used in anti-oxidant, anti-viral, anti-bacterial, anticancer, anti-inflammatory and anti-allergic therapies ^[26-29]. Baicalein is present in root bark, seeds, and leaves of O. indicum^[30]. For isolating Baicalein from O. indicum leaves, numerous methods are reported that includes conventional solvent extraction, hot and cold percolation and high-speed counter-current chromatography^[31-33].

Although, there are various CEM available for the Baicalein, due to its exhaustive nature, time-consuming nature that directly affects the percent yield of the Baicalein extract and concentration of the pure Baicalein in the extract. Considering the importance of baicalein, there is a need to enhance the extraction efficiency and extraction yield like important parameters by using efficient N-CEM. Keeping in view to that, there is no such report or documentation available related to the comparative evaluation of extraction method, extraction percent yield, and concentration of pure Baicalein, present research work was carried out.

MATERIAL AND METHOD:

General:

The authenticated dried leaves of O. indicum were ground to a powder using a mixer grinder (Havells India Ltd. Delhi, India). Leaf powder was sifted in a sieve shaker (CIP Machineries, Ahmedabad, GJ, India) to select uniform particle size, leaf powder was passed through sieves of different sizes (12, 24, 45, 85 and 120 mesh, Swastika Electric and Scientific Works, Ambala, HR, India) for a period of 20 min. The leaf powder passed through 120 mesh sieves was collected and used for further extraction experiments. The standard Baicalein (purity 98% by HPLC) was purchased from Sigma–Aldrich (St-Louis, MO, USA). All solvents used for the extraction and the chromatographic purpose were of analytical grade (Finar Chemicals Ltd., Ahmedabad, GJ, India) and HPLC grade (MERCK Specialties Pvt. Ltd. Mumbai, MH, India) respectively. Ultrasonic bath (Model: USB 6.5L (H), power: 230VPCi Analytics, Thane, MH, India) and Accelerated solvent extractor (E-914, Buchi India, Mumbai, MH, India) were used for the extraction purposes. The extracts were prepared freshly and stored in desiccators (Riviera Glass Pvt. Ltd., Mumbai, MH, India) under vacuum until the analysis.

Maceration:

Maceration is a commonly used conventional extraction technique widely used in the medicinal plant research. This method is the easiest and simple method of extraction. It involves the socking of the powdered plant material in a container containing solvent with proper stopper. The maceration extraction process was done by taking 60 grams of dried powdered leaves and subjected to macerate with Water, Ethanol: Water (50:50) and ethanol for 24 hrs. After 24 hrs the mixtures were further filtered concentrated using rotary vacuum evaporator and analyzed for Baicalein content by HPLC.

High-Performance Liquid Chromatography (HPLC) analysis:

All three extracts of O. indicum Linn macerated with Water, Ethanol: Water (50:50) and ethanol were analyzed using high-performance liquid chromatography (HPLC) at 278 nm. The chromatographic separation was performed on a C-18, 250 x 4.6 mm, 5um (X-Bridge, Waters) having oven temperature 40°C, with the mobile phase composed of 0.1% o-phosphoric acid in Water: Methanol (35:65 V/v) at a flow rate of 1.0 mL/min with total run time of 20 min.

Soxhlet assisted extraction technique (SAE):

SAE was used for the maximum recovery of Baicalein from the O. indicum leaves. 60 grams of powdered leaves were placed in porous bag or thimble made by using filter paper, which is placed in thimble chamber (Borosil, Mumbai, MH, India), which was inserted into a Soxhlet apparatus and extracted with 180 ml ethanol as an extraction solvent. The extraction solvent is heated in the round bottom flask and vaporizes into the thimble chamber and lastly condensed into the condenser. When the condense vapors reach to the chilled siphon tube arm it gets converted into the liquid and drip back into the round bottom flask again and the process was performed continued for 5h. After the extraction process completion, the sample present in the round bottom flask was collected and concentrated using vacuum rotary evaporator and analyzed for Baicalein content by using HPLC.

Ultra-sonic assisted extraction technique (UAE):

UAE involves the application of high intensity, high-frequency sound waves and their interaction with materials. UAE works by using the ultrasounds ranging from the 20 kHz to 2000 kHz. 40 grams of dried fruits was mixed with 200 ml of ethanol in a beaker. The extraction of O. indicum powder was carried out by placing the beaker in an ultrasonic bath with the fixed power of 150W. The beaker was immersed in the ultrasonic bath and extracted for 30 min. The water in the ultrasonic bath was circulated at 25°C to avoid the overheating produce by the ultrasound waves. The sample was then collected and concentrated using rotary vacuum evaporator and analyzed for the content of Baicalein by using HPLC.

Accelerated solvent extraction technique (ASE):

ASE is an efficient extraction method compare to maceration and SAE as the use of a minimum solvent. ASE based on parameters like temperature, time, pressure and hold time.5 gm of dry powder of O. indicum was mixed with 115 gm of silica sand and placed into the stainless-steel extraction cell to prevent the sample from aggregating and block the system tubing. Packed ASE cell contains the layer of silica sand and powder mixture cellulose filter papers and sand layers. 97% Ethanol was used as an extraction solvent for the overall extraction process with a temperature range of 78°C at pressure 78 psi*. Two static cycles were carried out in the overall extraction process. After the extraction, the total solvent volume was collected in the vial and the extracts were concentrated using rotary vacuum evaporator and analyzed for the content of Baicalein by using HPLC.

Identification and quantification of Baicalein in extracts using HPLC:

Ethanolic Extracts of different samples of O. indicum Linn by Accelerated solvent extraction, Ultrasound-assisted extraction; Soxhlet Extraction and Maceration were analyzed using high-performance liquid chromatography (HPLC) at 278nm. The chromatographic separation was performed on a C-18, 250 x 4.6 mm, 5um (X-Bridge, Waters) having oven temperature 40 degree Celsius, with the mobile phase composed of 0.1% o-phosphoric acid in Water: Methanol (35:65 v/v) at a flow rate of 1.0 mL/min and total run time was 20 min.

RESULTS AND DISCUSSION:

Maceration:

It is a basic technique used for the extraction process; it softens and breaks the plant cell wall to release the phytochemicals into the extraction solvent. It is CME and involves the transfer of heat through the mechanism of convection and conduction and also the choice of solvents determines the type of compound extracted from the plant material. In this research work, Maceration extraction process was performed by using three different extraction solvents viz. water, ethanol: water (50:50), and ethanol. The extraction of O. indicum in ethanol as extracting solvent shows maximum yield. On the basis of a percent, extraction yield ethanol was optimized as the better extraction solvent. The percentage yield in each solvent was determined and showed in Table 1. The HPLC estimation of baicalein in O. indicum by using ethanol as extracting solvents was also shown the higher concentration of baicalein as compare to the other extracting solvents. For the further extraction methods which include Soxhlet assisted extraction (SAE), Ultrasound extraction (UAE) and Accelerated solvent extraction (ASE) ethanol were selected as extracting solvent.

Table 1: Percent yield of maceration extraction of O. indicum using different solvents

Sr. No.	Solvents used for Extraction (ml)	Percent yield (%)
1	Water	1.21
2	Ethanol: water (50:50)	2.01
3	Ethanol	2.94

Identification and quantification of Baicalein in extracts using HPLC:

Ethanolic Extracts of different samples of O. indicum Linn by Accelerated solvent extraction, Ultrasound assisted extraction; Soxhlet Extraction and Maceration were analyzed using high performance liquid chromatography (HPLC) at 278nm. The chromatographic separation was performed on a C-18, 250 x 4.6 mm, 5um (X-Bridge, Waters) having oven temperature 40 degree Celsius, with the mobile phase composed of 0.1% o-phosphoric acid in Water: Methanol (35:65 V/v) at a flow rate of 1.0 mL/min and total run time was 20 min.

Soxhlet assisted extraction technique (SAE):

This CME requires a small quantity of solvent compared to the maceration process. Various factors such as temperature, solvent- sample ratio etc. need to be considered for this method. Soxhlet assisted extraction of O. indicum powder was performed by using ethanol as extracting solvent which was pre-optimized extraction solvent by maceration process. Total percent yield after ethanolic extraction was found to be 5.05.

Ultra-sonic assisted extraction technique (UAE):

The UAE has received great interest to overcome the disadvantages of Maceration and SAE such as small extraction percent yield. UAE is work on the basis of formation of ultrasound waves which transferred throughout the extraction solvent to form a cavity in the form of bubbles. Due to the continuous circulation of extraction solvent from the bubble cavities, that improves the mass transfer rate of extraction solvent. Due to increase in the mass transfer rate, the fractures forms in the plant cell walls which will enhance the permeation rate and a large amount of solvent enters into the plant tissue to extract the therapeutically active phytochemicals. The choice of suitable solvents for the SAE is an important parameter because of the physical properties like polarity, viscosity, surface tension, and vapor pressure that enhances the cavitation mechanism in the UAE process. Other parameters like frequency and power also considered a major factor for the UAE process, because the power produces physical effects like turbulence. Considering the importance of this factors ethanol was selected as an extraction solvent. Ultrasonic assisted extraction of O. indicum powder was also performed by using ethanol as the pre-optimized extraction solvent. The total percent yield of ethanolic extract of O. indicum by UAE was found to be 6.75 which were better than SAE. Parameters like irradiation time, exposure to ultrasonic waves plays a major role to get the high percent yield.

Accelerated solvent extraction technique (ASE):

ASE is an automated rapid extraction technique that utilizes common solvents at elevated temperature and pressure and thereby increases the efficiency of extraction of various compounds. ASE is an efficient form of liquid solvent extraction compared to maceration and Soxhlet extraction as the method using a minimal amount of solvent. In this technique, there is control of temperature and pressure for each individual sample and requires less than an hour for extraction. Accelerated solvent extraction of O. indicum powder was performed by using pre-optimized ethanol solvent. The ASE shows higher percentage yield as compared to the other extraction methodologies. The total percent yield of ethanolic extract of O. indicum was found to be 8.21. Percent yield of extraction of O. indicum by various extraction techniques using ethanol were shown in Table2.

Table 2: Percent yield of extraction of O. indicum by various extraction techniques using ethanol

Sr. No.	Extraction technique	Percent yield (%)
1	SAE	5.05
2	UAE	6.75
3	ASE	8.21

High-Performance Liquid Chromatography (HPLC) analysis:

HPLC fingerprinting of ethanolic extract of O. indicum obtained from the Maceration, SAE, UAE, and ASE techniques was performed. Different calibration standards viz. 1, 2, 3, 4, 5 and 6 ng/ml of baicalein were prepared and six points standard calibration curve for baicalein was developed using pre-calibrated HPLC method. The regression equation for linearity was found to be y= 9.84052e-006x + 0.0388703 with R2 value 0.9998 which is nearer to 1 indicates the graph is linear. The curve of Area vs. Amount was plotted as shown in figure 2. After the development of HPLC method standard solution of baicalein, the solution was prepared like that, the final concentration lies in the range of calibration standards. The baicalein sample was injected and the method was run for 20 min. The standard peak of baicalein was obtained at the retention time of 5.31 as shown in figure 3. Similarly, the concentrations of ethanolic extracts of Baicalein obtained from Maceration, UAE, SAE, and ASE were prepared and injected and results were recorded in terms of area and retention time.

The HPLC fingerprints of each extract obtained were shown in figure 4, 5, 6 and 7. From the HPLC analysis, it was observed that the extract obtained from the ASE technique shows the highest amount of baicalein. The total baicalein concentration obtained was shown in table3.

Table 3: Total concentration of Baicalein obtained from the various extraction techniques’

Samples	Area	Retention time	Concentration of Baicalein (ng)
Standard Baicalein	3,05290	5.310	3.000
Accelerated solvent extraction	1,46556	5.313	1.481
Ultrasound assisted extraction	1,02015	5.317	1.043
Soxhlet assisted extraction	22,696	5.313	0.262
Maceration	9138	5.313	0.129

Figure 2: Calibration curve for Baicalein by HPLC

Figure 3: HPLC fingerprinting of standard Baicalein

Figure 4: HPLC fingerprinting of Accelerated Solvents Extraction of O. indicum leaves

Figure 5: HPLC fingerprinting of Ultrasound Assisted Extraction of O. indicum leaves

Figure 6: HPLC fingerprinting of Soxhlet Assisted Extraction of O. indicum leaves

Figure 7: HPLC fingerprinting of Maceration of O. indicum leaves

CONCLUSION:

For effective extraction of baicalein from O. indicum, Ultrasound Assisted Extraction and Accelerated Solvent Extraction methods have been developed. Ethanol was found to be a promising solvent for the extraction of baicalein by using various non-conventional methodologies. Accelerated Solvent Extraction methodology was found to be the promising methodology for the extraction of baicalein from O. indicum.

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Received on 27.11.2018 Modified on 20.12.2018

Research J. Pharm. and Tech. 2019; 12(4):1817-1822.

DOI: 10.5958/0974-360X.2019.00303.2